Synthetic fluorophores are important tools in chemistry and biology. One of the main applications is their use as molecular probes in biomolecular imaging (Lavis, L. D.; Raines, R. T. ACS Chem Biol 2008, 3, 142). The ideal fluorophore for applications in biomolecular imaging should fulfill at least the following five criteria: First, the fluorophore absorbs and admits light at long wavelengths, preferentially above 600 nm. This ensures minimal phototoxicity when exciting the fluorophore, reduces background from cellular autofluorescence and increases tissue penetration for in vivo applications. Second, the fluorophore should possess high photostability to avoid rapid bleaching in the course of an experiment. Third, the fluorophore should be very bright, that is it should possess high extinction coefficients and high quantum yields. Fourth, a derivatization of the fluorophore with (i) reactive groups such as activated esters, (ii) ligands that specifically bind to other (bio)molecules in vitro or in vivo or (iii) molecules that can control the fluorescence properties of the fluorophore should be possible. Fifth, the fluorophore should be membrane permeable and show minimal background binding to biomolecules and biomolecular structures. While numerous fluorophores exist that fulfill the first four criteria, the cyanine fluorophore Cy5 being an example, there are few fluorophores available that also fulfill the fifth criterion.
Recently, a new class of fluorophores have been introduced which are based on the rhodamine structure but in which the oxygen atom in the xanthene ring has been replaced by silicon (Si-rhodamine) or germanium (Ge-rhodamine); cf. FIG. 1 (Xiao, Y.; Fu, M. 2008; CN 1810812. Fu, M.; Xiao, Y.; Qian, X.; Zhao, D.; Xu, Y. Chem Commun (Camb) 2008, 1780. Nagano, T.; Urano, Y.; Koide, Y. 2010; WO 2010126077, p 35. Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. ACS Chem Biol 2011, 6, 600. Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. J Am Chem Soc 2011, 133, 5680. Egawa, T.; Koide, Y.; Hanaoka, K.; Komatsu, T.; Terai, T.; Nagano, T. Chem Commun (Camb) 2011, 47, 4162):

These fluorophores show a large bathochromic shift relative to regular rhodamine derivatives with excitation and emission wavelengths above 600 nm. At the same time, they have high solubility, are very bright and photostable. In addition, there have been reports on their use in biomolecular imaging (Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. ACS Chem Biol 2011, 6, 600. Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. J Am Chem Soc 2011, 133, 5680. Egawa, T.; Koide, Y.; Hanaoka, K.; Komatsu, T.; Terai, T.; Nagano, T. Chem Commun (Camb) 2011, 47, 4162. Egawa, T. et al., J. Am. Chem. Soc, Epub ahead of print, DOI: 10.1021/ja205809h).
The specific coupling of fluorophores to proteins in living cells is an important method in life sciences. Such a specific coupling of fluorophores can be achieved by expressing the protein of interest as a fusion protein with an additional polypeptide that mediates the labeling of the fusion protein with the fluorophore (for review on labelling methods cf. Hinner, M. J.; Johnsson, K. Curr Opin Biotechnol 2010, 21, 766). Numerous approaches exist for achieving such a specific labeling in vitro and in vivo. Examples for such tags are small peptides that tightly bind to other molecules, proteins that tightly bind to other molecules, proteins that undergo a covalent reaction with other molecules and peptides to which other molecules are coupled with the help of enzymes. Methods that have been shown of particular utility for the labeling of intracellular protein are the tetracysteine tag that binds to biarsenical fluorophores, the SNAP-tag that irreversibly reacts with benzylguanine (BG) derivatives (Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K. Nat Biotechnol 2003, 21, 86), the CLIP-tag that reacts with benzylcytosine derivatives, the Halo-tag that reacts with primary chlorides and dihydrofolate reductase that binds to trimethoprim derivatives (Hinner, M. J.; Johnsson, K. Curr Opin Biotechnol 2010, 21, 766). Alternatively, a specific fluorescence labeling of a protein of interest can be achieved through the incorporation of an unnatural, fluorescent amino acid (Liu, C. C.; Schultz, P. G. Annu Rev Biochem 2010, 79, 413). While numerous fluorophores with excitation and emission maxima below 600 nm have been selectively coupled to intracellular proteins using one of the methods described above, the coupling of fluorophores to proteins with excitation and emission maxima above 600 nm remains problematic due to the membrane impermeability of such fluorophores and usually requires the introduction of the fluorophore into the cell through invasive methods such as microinjection (Keppler, A.; Arrivoli, C.; Sironi, L.; Ellenberg, J. Biotechniques 2006, 41, 167), bead-loading (Maurel, D.; Banala, S.; Laroche, T.; Johnsson, K. ACS Chem Biol 2010, 5, 507) or electroporation (Jones, S. A.; Shim, S. H.; He, J.; Zhuang, X. Nat Methods 2011, 8, 499).